How to map a plasmid by agarose gel bands?

[jinbtown]

I have a miniprep pGEX-6P-3 plasmid with a 0.75 kb insert. The insert was cloned into the vector with EcoR1 and Sal1 restriction endonucleases. When I run miniprep DNA from this clone and cut it with either only EcoR1 or Sal1, expecting one band at 5650 bp (pGEX is 4900 bp long), I come out with a band at 1500 bp as well (double the length of the insert). When I do a double digest, I get a band at 4900 bp as well as a brighter than expected band at 750 bp. Does this mean my insert has an internal EcoR1 or Sal1 site? Or both? I'm reading up on plasmid mapping but I'm not sure how to make sense of this.

The plasmid obviously has more than one site for each RE, because there isn't a single band for each singly cut plasmid. But I'm confused how to map this where these would make sense

EcoR1: 5.65 kb, 1.5 kb
Sal1: 5.65 kb, 1.5 kb
double digest: 4.9 kb, brighter than expected 0.75 kb

my first instinct was

-----EcoR1---0.75 kb----Sal1----0.75 kb----EcoR1----0.75 kb----Sal1

but I can't see how that would generate a band 5.65 bp when a single digest is run. It does explain the 1500 bp band though.

likewise, it explains the 0.75 kb bright band as well as the 4900 original vector band.

any and all help is appreciated.

[mchlbrmn]

I don't understand your confusion (or maybe I'm not seeing something right). It seems to me that your first instinct was right on. Look at it again: the triple insert explains both digests, doesn't it? The single enzyme digest can't cut off the insert at both sides, so one insert is left attached to the vector.

[jinbtown]

I don't understand your confusion (or maybe I'm not seeing something right). It seems to me that your first instinct was right on. Look at it again: the triple insert explains both digests, doesn't it? The single enzyme digest can't cut off the insert at both sides, so one insert is left attached to the vector.

man, it's just plain too late for this kind of work.
yes, that makes perfect sense. the EcoR1 single digest will leave the third insert attached, vice versa for the Sal1 digest. Of course that makes perfect sense. Then the double digest creates a triplet of inserts leading to a bright band. I don't know why I stared at this on paper for 30 minutes before checking here and going "oooohhhhhhhh"

much appreciated

[ladyefka]

Hello, I have some "plasmid troubles" as well. Although mine is not exactly the same, I didn't want to start new topic and hope I can get an answer here.

In one of my courses we I did the following:
- isolation of plasmid (pGem-T Vector, 3000bp) apparently containing an insert
- PCR using pUC/M13 Reverse Sequiencing Primer (binding site 161 - 177) and pUC/M13F (binding site 2941-2957)
- electrophoresis of PCR product.

I got a band, which should about 600bp (as a marker we used GeneRuler Ladder 100bp).

All I have is the information above and a sequence of multiple cloning site. My task is to say whether there was an insert in the plasmid and if so, what was its size.

I got pretty stuck trying to figure out which primer binds to which strand (don't know if it's important at all). It's been my nightmare for couple of days, so I would be glad for any kind of help. Thank you very much!

[mchlbrmn]

Do you actually have a 600 bp band, or you have a problem telling you that a 600 bp band is generated by PCR?
I Googled the vector, and it's 3kb w/o insert.
I'm don't know if the primer positions you give are with or w/o insert. OK, I looked that up too in a manual, and you gave the positions without any insert. Judging by the other primers I saw on a map, the positions they give are regardless of the fact that the second primer is actually a reverse primer, they didn't reverse the positions of start and stop to show this.
So, find the size of the PCR product with vector only, and subtract this from the PCR product size to find the insert size. To find the vector only size, since you are going around the corner of the 3000 / 1 numbering origin, you can add up the size of M13rev - 3000bp positions, (3000-2940), + the 177bp furthest position on the other side of the zero position. Understand?

I think your nightmare revolved around the fact that by the positions they give for both primers, it looks like they are both oriented forwards, and could not make a PCR product. However, on the diagram of the thing, the SP6 and T7 primers are shown facing toward each other, while in the text they are also indicated the same as the M13 primers, as if they are both forward primers. So, the text shows the position of the primer, but this does not indicate the orientation. Just assume that if they are facing each other to function in PCR, that you calculate that the PCR product extends from the lowest number (well, it's highest since the cloning site crosses the zero position. Maybe that was the other source of confusion) to the highest number.

[CrowSan]

The primers are actually close to each other (~ 160 bp apart as vector = 1-3000 and as plasmids are circular the numbering re-starts once the 3000 bp point is passed).

Without knowing the primer orientation (but assuming they bind to opposite strands!) then, if there is no insert:
a) if primers are orientated towards each other (across the cloning site) => 160 pb (the distance between the primers on the vector itself)

b) primers are orientated outwards from each other (pointing away from the cloning site) => 2,840 bp (vector size minus 160 pb distance between primers).

If any other (discrete) band is generated then the vector contains insert. In which case just subtract the "vector only" expected PCR product from the PCR size to determine the insert size.

It is desirable to know the orientation of the primers though as there are instances when an insert is present but would
still give a fragment similar to case b) above (i.e. primers face each other across the multiple cloning site (MCS) and the insert is actually 2.6 kb, so you would get 160bp (primer distance apart) + 2.6 kb insert = 2.8 kb).

Does this make any kind of sense or has senility finally struck me down....?
CrSn

[CrowSan]

Actually the 160 bp would be ~ 180-290 bp (as mchlbrmn indicated as the size would be distance + length of the primers)

[minman]

It appears that you have cloned 2 copies of your insert.

[Huh]

Lot's of good suggestions and ideas. But why don't you just sequence the darn thing and find out exactly what you have? Then you wouldn't have to guess. It's about $6 per reaction, sequence forward and reverse, that's $12 to figure out what's going on. I think it may be worth it.